CN110108825A - The method for building up and its finger-print of Lanqin oral liquid finger-print and application - Google Patents

Abstract

The invention discloses a method for establishing the fingerprint of Lanqin Oral Liquid, its fingerprint and its application. The present invention prepares the need testing solution with Lanqin Oral Liquid, with dissolving adenosine, epichun, cortex hydrochloride, chlorogenic acid, magnolialine, geniposide, benzoic acid, berberine hydrochloride, baicalin The solution of glycoside, wogonoside, baicalein and wogonin was used as the reference solution, and the liquid chromatogram was measured by high-performance liquid chromatography; the obtained liquid chromatogram was introduced into the similarity evaluation system of traditional Chinese medicine chromatographic fingerprints for analysis, and after multi-point Calibration, data matching, and analysis to obtain the fingerprint of Lanqin Oral Liquid. The method for establishing the fingerprint of the Lanqin oral liquid and the fingerprint thereof of the present invention can comprehensively reflect the quality information of the Lanqin oral liquid and ensure uniform and stable product quality.

Description

The establishment method of the fingerprint of Lanqin Oral Liquid, its fingerprint and its application

technical field

The invention relates to the field of drug analysis, in particular to a method for establishing fingerprints of Lanqin oral liquid, fingerprints and applications thereof.

Background technique

Lanqin Oral Liquid is a pure Chinese medicine preparation independently developed by Yangzijiang Pharmaceutical Group based on clinically proven prescriptions. Heat, analgesia, anti-inflammatory, antibacterial, antiviral five-in-one effect, mainly used for acute pharyngitis, sore throat, dry throat, burning throat caused by lung and stomach excess heat syndrome, upper respiratory tract infection, etc. It has obvious antiviral, antibacterial and anti-inflammatory effects, especially in reducing fever, eliminating sore throat, cough and other symptoms, with definite curative effect. Because of its wide curative effect, few adverse reactions, and a wide range of users, Lanqin Oral Liquid has attracted more and more attention from the society, and its development prospects are very broad.

The quality of medicines is the prerequisite to ensure the safety and effectiveness of clinical medicines. Currently, the method commonly used to evaluate the quality of Lanqin Oral Liquid preparations is to control the content of geniposide, but this method cannot reflect the overall curative effect of traditional Chinese medicine. To control the quality of Chinese patent medicines, it is necessary to control the substance group as a whole.

Contents of the invention

The following is an overview of the topics described in detail in this article. This summary is not intended to limit the scope of the claims.

The purpose of the present invention is to provide a method for establishing the fingerprint of Lanqin Oral Liquid and its fingerprint and application. By this method, Lanqin Oral Liquid and its key process intermediates include concentration process intermediate I and paste collection process intermediates. II. The fingerprints of the initial preparation process intermediate III and the fine preparation process intermediate IV, and qualitative and quantitative analysis of multiple medicinal ingredients in Lanqin Oral Liquid and its key process intermediates, according to different processes The changing law of content in the process and the quality control of different process links of the preparation provide a certain theoretical basis and support for the establishment of the whole process quality control system, which can comprehensively reflect the quality information of Lanqin Oral Liquid and ensure uniform product quality Stablize.

In an embodiment of the present invention, the present invention provides a method for establishing the fingerprint of Lanqin Oral Liquid, the method for establishing comprises the following steps:

(1) Preparation of the test solution: prepare the test solution with Lanqin Oral Liquid;

(2) Preparation of reference substance solution: with dissolved adenosine reference substance, Yichun reference substance, cortex base hydrochloride reference substance, chlorogenic acid reference substance, magnolialine reference substance, geniposide reference substance, benzoic acid The solutions of reference substance, berberine hydrochloride reference substance, baicalin reference substance, wogonin reference substance, baicalein reference substance and wogonin reference substance are reference substance solutions;

(3) adopt HPLC to measure the reference substance solution of the need testing solution of step (1) and step (2) respectively, and obtain the liquid chromatography of described need testing solution and described reference substance solution respectively;

(4) Import the liquid chromatogram obtained in step (3) into the Chinese medicine chromatographic fingerprint similarity evaluation system for analysis, and obtain the fingerprint of Lanqin Oral Liquid.

In an embodiment of the present invention, the Lanqin Oral Liquid described in step (1) can be replaced by the key process intermediate of Lanqin Oral Liquid; the key process intermediate of Lanqin Oral Liquid includes concentrated process intermediate I, extract extract Process intermediate II, initial formulation process intermediate III, fine formulation process intermediate IV; moreover, the samples of Lanqin Oral Liquid and its key process intermediates come from different batches of products produced.

The preparation process of Lanqin Oral Liquid is as follows: Take 300.0g of Radix Radix, 240.0g of Scutellaria, 300.0g of Gardenia, 120.0g of Cortex Phellodendron, 100.0g of Panda Dahai, the above five flavors, add water and decoct three times, the first time for 2 hours, the second time, 1 hour each for the third time. Combine the decoctions, filter, and concentrate the filtrate to a relative density of 1.10-1.12 (70°C). Add ethanol to make the alcohol content reach about 60%, let stand for 12 hours, and filter. Stand below 10°C for 24 hours, filter, fill, and sterilize. Among them, "the filtrate is concentrated to a relative density of 1.10-1.12 (70°C)" is the concentration process intermediate; "the filtrate is decompressed to recover ethanol and concentrated to a relative density of 1.15-1.20 (60°C)" is the cream collection process intermediate II, "Take another 300g of sucrose to make syrup, combine with the above liquid medicine, heat and boil for 15 minutes, add 3g of sodium benzoate," is the initial preparation process intermediate III; "0.3% polysorbate 80, add water to 1000ml, mix well ” is the fine compound process intermediate IV.

In the embodiment of the method for establishing the fingerprint of Lanqin Oral Liquid of the present invention, wherein, the preparation of the test solution in the step (1) includes: accurately drawing 100-300 μl of Lanqin Oral Liquid in a 10mL centrifuge tube, Add 4-8 mL of methanol aqueous solution with a volume fraction of 50%-100% for dilution, mix well and pass through a 0.45 μm microporous membrane. As a preferred solution, the preparation of the test solution in the step (1) includes: accurately absorbing 200 μl of Lanqin Oral Liquid into a 10 mL centrifuge tube, adding 4.8 mL of methanol to dilute, and passing through a 0.45 μm microporous membrane after mixing.

In the embodiment of the method for establishing the fingerprint of Lanqin Oral Liquid of the present invention, wherein, the preparation of the reference substance solution in the step (2) includes: accurately weighing the adenosine reference substance, the Yichun reference substance, hydrochloric acid Cortex base reference substance, chlorogenic acid reference substance, magnolialine reference substance, geniposide reference substance, benzoic acid reference substance, berberine hydrochloride reference substance, baicalin reference substance, wogonin reference substance, baicalein reference substance and wogonin reference substance, add methanol aqueous solution with a volume fraction of 70%-100% to make each 1mL contain 5-8 μg adenosine, 2-6 μg echinacea, 5-8 μg cortexine hydrochloride, 30-50 μg chlorogenic acid, 2-5 μg magnolialine, 200-300 μg geniposide, 100-200 μg benzoic acid, 2-5 μg berberine hydrochloride, 150-250 μg baicalin, 50-100 μg wogonin, 8-15 μg baicalein, 8 ~15 μg wogonin reference substance solution. As a preferred solution, the preparation of the reference substance solution in the step (2) includes: taking an appropriate amount of the baicalin reference substance, accurately weighing it, adding a volume fraction of 70% methanol aqueous solution to dissolve, and preparing a reference substance with a content of 208 μg·mL ⁻¹ Solution, respectively take adenosine reference substance, Yichun reference substance, Phellodendri hydrochloride reference substance, chlorogenic acid reference substance, magnolialine reference substance, geniposide reference substance, benzoic acid reference substance, berberine hydrochloride reference substance Proper amount of baicalin reference substance, baicalein reference substance and wogonin reference substance were accurately weighed, dissolved in methanol, and the contents were prepared to be 7.52μg·mL ^-1 , 3.84μg·mL ^-1 , 6.44μg·mL -1 , respectively. mL ^-1 , 32.96μg·mL ^-1 , 2.52μg·mL ^-1 , 284.00μg·mL ^-1 , 162.00μg·mL ^-1 , 4.56μg·mL ^-1 , 98.00μg·mL ^-1 , 10.60μg·mL ^-1 and 9.76μg·mL ^-1 of the reference solution.

In the embodiment of the method for establishing the fingerprint of Lanqin Oral Liquid of the present invention, wherein, the step (3) includes setting HPLC chromatographic conditions: the chromatographic column adopts octadecylsilane bonded silica gel as filler, and the column temperature is 25- 35°C, the flow rate is 0.8-1.2mL/min, the detection wavelength is 250-320nm, and DAD detection is turned on; acetonitrile is used as mobile phase A, and phosphoric acid aqueous solution with a volume fraction of 0.06-0.2% is used as mobile phase B. Gradient elution in the range of ～95:100～0;

Accurately draw 5-20 μL of the test solution and the reference solution respectively, inject them into a high-performance liquid chromatograph, measure, and obtain the liquid chromatography of the test solution and the reference solution respectively.

As a preferred version, the step (3) includes setting the HPLC chromatographic conditions: the chromatographic column adopts octadecylsilane bonded silica gel as a filler, with Waters 2695ACQUITY HPLC ^TM (4.6mm × 250mm, 5.0 μm) as a chromatographic column, the column temperature 30°C, the flow rate is 1.0mL/min, the detection wavelength is 230nm, and DAD detection is turned on; acetonitrile is used as mobile phase A, and 0.2% phosphoric acid aqueous solution is used as mobile phase B to perform gradient elution. Mobile phases A and B The ratio change of A: B is 0～12min, A: B is 5%:95%→11%:89%; 12～24min, A:B is 11%:89%→11%:89%; 24～38min, A: B is 11%: 89% → 23%: 77%; 38-50min, A: B is 23%: 77% → 28%: 72%; 50-55min, A: B is 28%: 72% → 31% : 69%; 55～70min, A: B is 31%: 69% → 100%: 0%;

Accurately draw 10 μL of the test solution and the reference solution respectively, inject it into a high-performance liquid chromatograph, measure, and obtain the liquid chromatography of the test solution and the reference solution respectively.

In the embodiment of the method for establishing the fingerprint of Lanqin Oral Liquid of the present invention, wherein, the Chinese medicine chromatographic fingerprint similarity evaluation system 2004A edition that described step (4) adopts, described step (4) comprises: the step ( 3) The obtained liquid chromatography data import, multi-point calibration, data matching, analysis to obtain the fingerprint of Lanqin Oral Liquid.

In the embodiment of the method for establishing the fingerprint of Lanqin Oral Liquid of the present invention, wherein, the fingerprint of Lanqin Oral Liquid obtained in the step (4) includes peak No. 1 corresponding to adenosine and peak 4 corresponding to Yichun. Peak No. 5, peak No. 5 corresponding to cortex base hydrochloride, peak No. 6 corresponding to chlorogenic acid, peak No. 7 corresponding to magnolialine, peak No. 9 corresponding to geniposide, peak No. 15 corresponding to benzoic acid, berberis hydrochloride Peak 16 corresponding to base, peak 17 corresponding to baicalin, peak 22 corresponding to wogonin, peak 24 corresponding to baicalein, peak 25 corresponding to wogonin, the relative retention times are 0.120 and 0.587 respectively , 0.711, 0.777, 0.806, 1.000, 1.882, 1.946, 2.002, 2.615, 2.856, 3.003.

In the embodiment of the method for establishing the fingerprint of Lanqin oral liquid of the present invention, wherein, the common peaks of the fingerprint of Lanqin oral liquid obtained in the step (4) also include No. 2 peaks with a relative retention time of 0.382, relative Peak No. 3 with a retention time of 0.453, peak No. 8 with a relative retention time of 0.846, peak No. 10 with a relative retention time of 1.400, peak No. 11 with a relative retention time of 1.499, and peak No. 12 with a relative retention time of 1.731. Peak No. 13 with a retention time of 1.812, peak No. 14 with a relative retention time of 1.847, peak No. 18 with a relative retention time of 2.227, peak No. 19 with a relative retention time of 2.375, and peak No. 20 with a relative retention time of 2.414. Peak No. 21 with a retention time of 2.495, peak No. 23 with a relative retention time of 2.835, and peak No. 26 with a relative retention time of 3.015.

In the embodiment of the method for establishing the fingerprint of Lanqin Oral Liquid of the present invention, wherein, the fingerprint of Lanqin Oral Liquid obtained in the step (4) is shown in FIG. 2 .

In the second aspect, the present invention provides a method for establishing the fingerprint of the above-mentioned Lanqin Oral Liquid and its key process intermediates in the establishment of the fingerprints include the concentration process intermediate I, the cream collection process intermediate II, the initial preparation process intermediate III, The application of the fingerprint of the fine preparation process intermediate IV, or the establishment method of the fingerprint of the above-mentioned Lanqin Oral Liquid and its fingerprint in Lanqin Oral Liquid and its key process intermediates, including the concentration process intermediate I and the extract process intermediate II. The application of qualitative and quantitative analysis of multiple active components of multiple medicinal materials in the preliminary preparation process intermediate III and fine preparation process intermediate IV, or the establishment method of the fingerprint of the above-mentioned Lanqin oral liquid and its fingerprint in different preparations The application of quality control in process links.

The technical effects achieved by the present invention include at least one or more of the following:

(1) The fingerprints of Lanqin Oral Liquid obtained from different batches of Lanqin Oral Liquid can represent most of the efficacy of Lanqin Oral Liquid, and are more effective in characterizing the quality of Lanqin Oral Liquid.

(2) Treat the fingerprint spectrum of Lanqin Oral Liquid as a whole, pay attention to the fingerprint characteristic peaks and mutual relations of various chemical components, pay attention to the overall appearance characteristics of Lanqin Oral Liquid, and avoid measuring only one or two index chemical components The one-sidedness of judging the quality of Lanqin Oral Liquid reduces the possibility of errors caused by human subjective judgment.

(3) Based on the fingerprint of Lanqin Oral Liquid, the key process intermediates of Lanqin Oral Liquid were established, including Concentration Process Intermediate I, Cream Collection Process Intermediate II, Initial Preparation Process Intermediate III, and Fine Formulation Process Intermediate IV fingerprints, and qualitative and quantitative analysis of multiple effective components of multiple medicinal materials in each process step, according to the trend and degree of content change, confirm the key process control links, and formulate corresponding key point quality control standards, which can be used for The production process monitoring of Lanqin Oral Liquid provides a certain theoretical basis and support for the establishment of the whole process quality control system to ensure the safety and effectiveness of clinical medication.

(4) In the prior art, methanol-water, acetonitrile-water, methanol-0.2% phosphoric acid water, etc. are used for gradient elution in the separation and content determination of single medicinal materials in Lanqin Oral Liquid. The present invention uses acetonitrile-0.2% Gradient elution with phosphoric acid water. By examining the elution effects of these four systems, comparing the chromatograms (Fig. 3-Fig. 6), it can be seen that in the chromatograms eluted by methanol-water and methanol-0.2% phosphoric acid water systems, the number of chromatographic peaks is less, which is not enough It fully represents the material basis of Lanqin Oral Liquid; although the number of chromatographic peaks in the chromatogram eluted by the acetonitrile-water system is similar to that of acetonitrile-0.2% phosphoric acid water, the resolution of the peaks is poor. The present invention uses acetonitrile-0.2% phosphoric acid aqueous solution as the eluting solvent, and the chromatographic peaks obtained under this condition are significantly better than other eluting systems.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention may be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.

Description of drawings

The accompanying drawings are used to provide a further understanding of the technical solution of the present invention, and constitute a part of the description, and are used together with the embodiments of the application to explain the technical solution of the present invention, and do not constitute a limitation to the technical solution of the present invention.

Fig. 1 is the HPLC chromatogram of 12 kinds of reference substances;

In Figure 1, 1: adenosine reference substance, 2: Yichun reference substance, 3: Phellodendri hydrochloride reference substance, 4: chlorogenic acid reference substance, 5: magnolialine reference substance, 6: geniposide reference substance 7: benzoic acid reference substance, 8: berberine hydrochloride reference substance, 9: baicalin reference substance, 10: wogonin reference substance, 11: baicalein reference substance, 12: wogonin reference substance;

Fig. 2 is 19 batches of Lanqin Oral Liquid Fingerprint Spectrum Overlay Spectrum Spectra in Example 1 of the present invention;

Fig. 3 is the common pattern collection of illustrative plates of the Lanqin oral liquid measured in the embodiment of the present invention 1;

In Figure 3, peak No. 1: adenosine, peak No. 4: Yichun, peak No. 5: cortexine hydrochloride, peak No. 6: chlorogenic acid, peak No. 7: magnolialine, peak No. 9: gardenia Glycosides, peak 15: benzoic acid, peak 16: berberine hydrochloride, peak 17: baicalin, peak 22: wogonin, peak 24: baicalein, peak 25: wogonin;

Fig. 4 is the chromatogram of Lanqin Oral Liquid measured by methanol-0.2% phosphoric acid water gradient elution;

Fig. 5 is the chromatogram of Lanqin Oral Liquid measured by methanol-water gradient elution;

Fig. 6 is the chromatogram of Lanqin Oral Liquid measured by acetonitrile-water gradient elution;

Fig. 7 is the positive control chromatogram of Radix Radix Radix medicinal material positive control in the embodiment 1 of the present invention;

Fig. 8 is the chromatographic comparison chart of positive control chromatogram and fingerprint of Radix Radix Radix medicinal material positive control chromatogram in the embodiment 1 of the present invention;

Fig. 9 is the negative control chromatogram of Radix isatidis medicinal material in Example 1 of the present invention;

Fig. 10 is a chromatogram comparison chart of the negative control chromatogram and fingerprint of Radix isatidis medicinal material in Example 1 of the present invention;

Fig. 11 is the positive control chromatogram of Scutellaria baicalensis medicinal material in Example 1 of the present invention;

Fig. 12 is the chromatographic comparison chart of positive control chromatogram and fingerprint of Scutellaria baicalensis medicinal material in Example 1 of the present invention;

Fig. 13 is the negative control chromatogram of Scutellaria baicalensis medicinal material in Example 1 of the present invention;

Fig. 14 is the chromatographic comparison chart of negative control chromatogram and fingerprint of Scutellaria baicalensis medicinal material in Example 1 of the present invention;

Fig. 15 is the positive control chromatogram of gardenia medicinal material in Example 1 of the present invention;

Fig. 16 is the chromatogram comparison chart of positive control chromatogram and fingerprint of gardenia medicinal material in Example 1 of the present invention;

Fig. 17 is the negative control chromatogram of gardenia medicinal material in Example 1 of the present invention;

Fig. 18 is a chromatographic comparison chart of the negative control chromatogram and fingerprint of gardenia medicinal material in Example 1 of the present invention;

Fig. 19 is the positive control chromatogram of Phellodendron Phellodendri medicinal material in Example 1 of the present invention;

Fig. 20 is the chromatogram comparison chart of positive control chromatogram and fingerprint of Cortex Phellodendri in Example 1 of the present invention;

Fig. 21 is the negative control chromatogram of Phellodendron Phellodendri medicinal material in Example 1 of the present invention;

Fig. 22 is the chromatographic comparison chart of the negative control chromatogram and fingerprint of Phellodendron Phellodendri in Example 1 of the present invention;

Fig. 23 is the superimposed spectrum of fingerprints of 9 batches of Lanqin concentration process intermediate I in Example 3 of the present invention;

Figure 24 is the common pattern spectrum of 9 batches of Lanqin concentration process intermediate I in Example 3 of the present invention;

Fig. 25 is the superimposed spectrum of fingerprints of 9 batches of blue qin extract process intermediate II in Example 3 of the present invention;

Figure 26 is the common pattern spectrum of 9 batches of Lanqin Harvesting Process Intermediate II in Example 3 of the present invention;

Fig. 27 is the superimposed spectrum of fingerprints of 6 batches of Lanqin primary preparation process intermediate III in Example 3 of the present invention;

Fig. 28 is the common pattern spectrum of 6 batches of Lanqin primary preparation process intermediate III in Example 3 of the present invention;

Fig. 29 is the superimposed spectrum of fingerprints of 6 batches of Lanqin essence compounding process intermediate IV in Example 3 of the present invention;

Fig. 30 is the common pattern spectrum of 6 batches of Lanqin essence compounding process intermediate IV in Example 3 of the present invention;

Figures 31A-31C are the content changes of each index component in the preparation process of Lanqin in Example 4 of the present invention;

Fig. 32 is a component ratio diagram of the content change of each index component of the Lanqin process intermediate in Example 4 of the present invention.

Detailed ways

In order to make the purpose, technical solution and advantages of the present application clearer, the embodiments of the present invention will be described in detail below. It should be noted that, in the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined arbitrarily with each other.

The instruments and reagents used in the examples are as follows:

1. Instruments and reagents

1.1 Instrument

One hundred thousandth balance (Sartorius BT125D, Sartorius Scientific Instrument Co., Ltd.); desktop high-speed centrifuge (TG16-WS, Changsha Xiangyi Centrifuge Instrument Co., Ltd.); high performance liquid chromatography (LC-10A, Japan Island Jin company, DAD detector); ultrasonic cleaner (KH3200B, Kunshan Hechuang Ultrasonic Instrument Co., Ltd.); traditional Chinese medicine pulverizer (HC-150, Huangcheng high-speed multifunctional pulverizer model).

1.2 Reagent

Adenosine reference substance (batch number: 161207), Biaobaoyichun reference substance (batch number: 170420), Phellodendri hydrochloride reference substance (batch number: 170307), chlorogenic acid reference substance (batch number: 160507), magnolialine reference substance ( Batch number: 170104), geniposide reference substance (batch number: 170112), benzoic acid reference substance (batch number: 170507), berberine hydrochloride reference substance (batch number: 161223), baicalin reference substance (batch number: 170216), Scutellaria baicalensis Glycoside reference substance (batch number: 170109), baicalein (reference substance batch number: 170324), wogonin reference substance (batch number: 170320), the above standard substances were all purchased from Shanghai Ronghe Pharmaceutical Technology Co., Ltd., with a content of ≥98%.

Chromatographic methanol and acetonitrile were purchased from Sigma Company of the United States, and chromatographic phosphoric acid was purchased from Tianjin Kemiou Chemical Reagent Co., Ltd. The water used was Watsons distilled water.

A total of 19 batches of Lanqin Oral Liquid samples were collected, all produced by Yangzijiang Pharmaceutical Group Co., Ltd. The basic information of 19 batches of Lanqin Oral Liquid samples is shown in Table 1.

Table 1 Basic information of samples of Lanqin Oral Liquid

A total of 9 batches of samples of Lanqin Concentration Process Intermediate I were collected, all of which were produced by Yangzijiang Pharmaceutical Group Co., Ltd. See Table 2 for the basic information of 9 batches of Sapphire Concentration Process Intermediate I.

Table 2 The basic information of the samples for the concentration process intermediate I of Lanqin

A total of 9 batches of samples of Lanqin Harvesting Process Intermediate II were collected, all of which were produced by Yangzijiang Pharmaceutical Group Co., Ltd. See Table 3 for the basic information of 9 batches of Lanqin Harvesting Process Intermediate II for the samples.

Table 3 The basic information of the sample of Lanqin Shou Gao Process Intermediate II

A total of 6 batches of intermediate III samples of Lanqin primary preparation process were collected, all of which were produced by Yangzijiang Pharmaceutical Group Co., Ltd. See Table 4 for the basic information of the 6 batches of Lanqin Primary Compounding Process Intermediate III for the samples.

Table 4 The basic information of the samples for the preparation of Lanqin primary preparation process intermediate III

A total of 6 batches of samples of Lanqinjing Compounding Process Intermediate IV were collected, all of which were produced by Yangzijiang Pharmaceutical Group Co., Ltd. See Table 5 for the basic information of the 6 batches of Lanqin primary preparation process intermediate IV for the samples.

Table 5 The basic information of the samples for the initial preparation of Lanqin, the process intermediate IV

Example 1 The establishment method and methodological investigation of the fingerprint of Lanqin Oral Liquid

1. Method for establishing the fingerprint of Lanqin Oral Liquid

The method for establishing the fingerprint of Lanqin Oral Liquid comprises the following steps:

(1) Preparation of the test solution: take 19 different batches of Lanqin oral liquid in the above table 1, accurately draw 200 μl into a 10mL centrifuge tube, add 4.8mL of methanol to dilute, mix well and pass through a 0.45 μm micro Pore filter membrane, as need testing solution;

(2) Preparation of reference substance solution: take an appropriate amount of baicalin reference substance, accurately weighed, add 70% methanol to dissolve, and prepare a reference substance solution with a content of ²⁰⁸ μg mL Spring reference substance, phellodine hydrochloride reference substance, chlorogenic acid reference substance, magnolialine reference substance, geniposide reference substance, benzoic acid reference substance, berberine hydrochloride reference substance, wogonin reference substance, baicalein reference substance and wogonin reference substance, accurately weighed, dissolved in methanol, and prepared to have the contents of 7.52μg·mL ^-1 , 3.84μg·mL ^-1 , 6.44μg·mL ^-1 , 32.96μg·mL ^-1 , 2.52 Control substances of μg·mL ^-1 , 284.00 μg·mL ^-1 , 162.00 μg·mL ^-1 , 4.56 μg·mL ^-1 , 98.00 μg·mL ^-1 , 10.60 μg·mL ^-1 , 9.76 μg·mL ^-1 solution.

(3) HPLC chromatographic conditions: the chromatographic column uses octadecylsilane bonded silica gel as the filler, Waters 2695ACQUITY HPLC ^TM (4.6mm×250mm, 5.0μm) as the chromatographic column, the column temperature is 30°C, and the flow rate is 1.0mL/min , the detection wavelength is 230nm, and DAD detection is turned on. Use acetonitrile as mobile phase A and 0.2% phosphoric acid aqueous solution as mobile phase B for gradient elution. The ratio of mobile phase A and B changes from 0 to 12 minutes, and the ratio of A:B is 5%:95%→11%. : 89%; 12-24min, A: B is 11%: 89%→11%: 89%; 24-38min, A: B is 11%: 89%→23%: 77%; 38-50min, A: B is 23%: 77% → 28%: 72%; 50 ~ 55min, A: B is 28%: 72% → 31%: 69%; 55 ~ 70min, A: B is 31%: 69% → 100% : 0%;

(4) Accurately draw 10 μL of the test solution and the reference solution respectively, inject them into a high-performance liquid chromatograph, measure, and obtain the liquid chromatography of the test solution and the reference solution respectively.

(5) Import the liquid chromatogram into the fingerprint similarity evaluation software (Chinese medicine chromatographic fingerprint similarity evaluation system version 2004A) for analysis, take 17050221 batches of oral liquid as the reference atlas, select the "time window width" as 0.1min, and use the average Mathematical calculation, multi-point calibration, data matching, generate overlay chromatogram (see Figure 3) and control spectrum (see Figure 2). The fingerprints of 19 batches of Lanqin Oral Liquid were analyzed for similarity with the generated control fingerprints, and the results are shown in Table 6.

Table 6 Similarity of fingerprints of 19 batches of Lanqin Oral Liquid

It can be seen from Table 6 that the similarity between the fingerprints of different batches of Lanqin Oral Liquid and the control spectrum is between 0.995 and 1, indicating that the similarity between different batches is high and the process is relatively stable.

Compared with 12 kinds of reference substances, it was confirmed that the peak at 2.58min was adenosine (peak No. 1), the peak at 12.56min was Biaoyichun (peak No. 4), the peak at 15.18min was cortexine hydrochloride (peak No. 5), and the peak at 16.68 The min peak is chlorogenic acid (peak No. 6), the peak at 17.23 min is magnolanine (peak No. 7), the peak at 21.53 min is geniposide (peak No. 9), and the peak at 40.13 min is benzoic acid (peak No. 15). The peak at 41.45min is berberine hydrochloride (peak No. 16), the peak at 47.43min is baicalin (peak No. 17), the peak at 55.69min is wogonin (peak No. 22), and the peak at 60.88min is baicalein (peak No. 24) , The peak at 64.01min is wogonin (peak No. 25). Among them, geniposide with better resolution, larger and stable peak area, and moderate retention time was selected as the reference peak (S). A total of 26 common peaks were identified in the HPLC fingerprint with geniposide as the reference peak.

2. Investigation of Fingerprint Methodology

2.1 Precision test

Get Lanqin Oral Liquid (17050221), prepare the test solution according to the preparation method of the test solution in Example 1, inject 6 needles continuously according to the chromatographic conditions in Example 1, and obtain 6 chromatograms. Taking the geniposide peak as the reference peak, the relative peak area and relative retention time of each common peak and the reference peak were calculated, and the RSD value was calculated. The results are shown in Table 7 and Table 8.

Table 7 Lanqin Oral Liquid Fingerprint Precision Test Investigation Results (relative peak area of each common peak)

Table 8 Lanqin Oral Liquid Fingerprint Precision Test Investigation Results (relative retention time of each common peak)

It can be seen from Table 7 and Table 8 that the relative peak area RSD% of each common chromatographic peak < 3%, and the relative retention time RSD% < 3%, indicating that the precision of the method is good.

2.2 Repeatability test

Get Lanqin Oral Liquid (17050221), weigh 6 parts in parallel, prepare 6 parts of need testing solution by the preparation method of the need testing solution of Example 1, inject samples respectively by the chromatographic conditions in Example 1, obtain 6 chromatograms picture. Taking the geniposide peak as the reference peak, the relative peak area and relative retention time of each common peak and the reference peak were calculated, and the RSD value was calculated. The results are shown in Table 9 and Table 10.

Table 9 Lanqin oral liquid fingerprint repeatability test results (relative peak area of each common peak)

Table 10 Lanqin oral liquid fingerprint repeatability test investigation results (relative retention time of each common peak)

It can be seen from Table 9 and Table 10 that the relative peak area RSD% of each common chromatographic peak < 3%, and the relative retention time RSD% < 3%, indicating that the method has good repeatability.

2.3 Stability test

Get Lanqin Oral Liquid (17050221), prepare 6 parts of need testing solution by the preparation method of need testing solution of embodiment 1, press chromatographic condition in embodiment 1, respectively in 0h, 2h, 4h, 8h, 12h, 24h Conditional injection, get 6 chromatograms. Taking the baicalin peak as the reference peak, the relative peak area and relative retention time of each common peak and the reference peak were calculated, and the RSD value was calculated. The results are shown in Table 11 and Table 12.

Table 11 Lanqin oral liquid fingerprint stability test results (relative peak area of each common peak)

Table 12 Lanqin Oral Liquid Fingerprint Stability Test Investigation Results (relative retention time of each common peak)

As can be seen from Table 11 and Table 12, the relative peak area RSD%<3% and the relative retention time RSD%<3% of each common chromatographic peak indicate that the test solution is basically stable within 24h.

3. Correlation between Lanqin Oral Liquid and various raw materials and common peak attribution analysis

The positive control chromatograms and negative control chromatograms of Radix Radix Radix Isatidis, Radix Astragali, Gardenia, Phellodendron Phellodendri in Oral Liquid were determined according to the method of Example 1, and the fingerprints of Lanqin Oral Liquid and the positive control chromatograms and negative control chromatograms of each medicinal material were detected by DAD detector. Analyze the ultraviolet absorption of the chromatographic peaks in the chromatogram, compare the retention time of the chromatographic peaks, and finally confirm the attribution peaks of the common chromatographic peaks in the fingerprint of Lanqin Oral Liquid on the medicinal material chromatogram.

3.1 Analysis of common peaks in Radix isatidis

See Figure 4 and Figure 5 for the positive control chromatogram of Radix Radix Radix and the chromatographic comparison between the positive control chromatogram and the fingerprint. See Figure 6 and Figure 7 for the chromatogram of the negative control chromatogram of Radix isatidis and the chromatographic comparison between the chromatogram of the negative control chromatogram and the fingerprint. Through the comparison of the chromatograms, it can be seen that peaks 1 and 4 in the common peaks of the fingerprints come from Radix Radix Radix.

3.2 Analysis of common peaks in Scutellaria baicalensis

See Figure 8 and Figure 9 for the positive control chromatogram of Scutellaria baicalensis and the chromatographic comparison between the positive control chromatogram and the fingerprint. See Figure 10 and Figure 11 for the negative control chromatogram of Scutellaria baicalensis and the chromatographic comparison between the negative control chromatogram and the fingerprint. Through the comparison of the chromatograms, it can be seen that peaks 12, 13, 14, 18, 19, 20, 21, 22, 23, 24, 25, and 26 in the common peaks of the fingerprints come from Scutellaria baicalensis.

3.3 Analysis of common peaks in Gardenia jasminoides

See Figure 12 and Figure 13 for the positive control chromatogram of Gardenia medicinal material and the chromatogram comparison between the positive control chromatogram and the fingerprint. See Figure 14 and Figure 15 for the negative control chromatogram of Gardenia medicinal material and the chromatographic comparison between the negative control chromatogram and the fingerprint. Through the comparison of the chromatograms, it can be known that peaks 2, 3, 6, 9, 13, 14, 15, and 17 in the common peaks of the fingerprints come from gardenia medicinal materials.

3.4 Analysis of common peaks in Cortex Phellodendri

See Figure 16 and Figure 17 for the positive control chromatogram of Cortex Phellodendri and the chromatographic comparison between the positive control chromatogram and the fingerprint. See Figure 18 and Figure 19 for the negative control chromatogram of Cortex Phellodendri and the chromatographic comparison between the negative control chromatogram and the fingerprint. Through the comparison of the chromatograms, it can be seen that peaks 5, 7, 8, 10, 11, and 16 in the common peaks of the fingerprints come from Phellodendron Phellodendri.

3.5 Analysis of the peaks shared by Lanqin Oral Liquid and various medicinal materials

From the analysis of the above results, it can be seen that the 26 common peaks in the fingerprint of Lanqin Oral Liquid basically find a clear attribution in the chromatograms of the positive control and negative control of the four medicinal materials of Radix Radix Isatidis, Scutellaria, Gardenia, and Phellodendron Phellodendri. It can be seen that peaks 1 and 4 in the common peaks of the fingerprints come from Radix Radix Radix; peaks 12, 18, 19, 20, 21, 22, 23, 24, 25, and 26 in the common peaks come from Scutellaria baicalensis; , Peaks 7, 8, 10, 11, and 16 come from Cortex Phellodendri; peaks 2, 3, 6, 9, 15, and 17 in the common peaks come from Gardenia; peaks 13 and 14 exist in Scutellaria baicalensis and Gardenia In the medicinal materials. It can be seen that the fingerprint of Lanqin Oral Liquid can basically represent the material basis of Lanqin Oral Liquid.

Example 2 Detection and Methodological Investigation of the Multi-wavelength Quantitative Components of the Lanqin Oral Liquid Fingerprint

1. Detection of multi-wavelength quantitative components

Adenosine, Yichun, phellodine hydrochloride, chlorogenic acid, magnolialine, geniposide, benzoic acid, berberine hydrochloride, wogonin, baicalein, wogonin 12 kinds of drug efficacy index ingredients, retained The times are: 2.58, 12.56, 15.18, 16.68, 17.23, 21.53, 40.13, 41.45, 47.43, 55.69, 60.88, 64.01min. Turn on the DAD detector for full-wavelength scanning, compare the peak areas of each drug efficacy index component under different wavelength conditions, and obtain the optimal detection wavelength of each index component. Acid, magnolialine, geniposide, benzoic acid and berberine hydrochloride have greater absorption at 230nm; baicalin, wogonoside, baicalein, wogonin have greater absorption at 276nm. Finally, select and record the retention time and peak area of adenosine, epicalyptus, cortexine hydrochloride, chlorogenic acid, magnolialine, geniposide, benzoic acid, and berberine hydrochloride under 230nm conditions, and calculate the peak area of each characteristic peak. RSD value: Record the retention time and peak area of baicalin, wogonin, baicalein and wogonin at 276nm, and calculate the RSD value of each characteristic peak.

2. Investigation on multi-wavelength quantitative component detection methodology

2.1 Linear inspection

Precisely measure an appropriate amount of baicalin reference solution, add 70% methanol to dilute it step by step, configure a series of concentration gradient reference solution, inject and detect under the established chromatographic conditions, measure and record the peak area at a wavelength of 276nm Precisely measure an appropriate amount of mixed reference substance solution, add methanol to dilute step by step, configure a series of mixed reference substance solutions with concentration gradients, inject samples for detection under established chromatographic conditions, measure and record peak areas at wavelengths of 230nm and 276nm Take peak area as ordinate (Y), concentration (X) as abscissa, obtain the regression equation of each index composition as shown in table 13:

Table 13 Regression equation of each efficacy index component

2.2 Precision test

Get Lanqin Oral Liquid (17050232), prepare the test solution according to the preparation method of the test solution in Example 1, inject 6 needles continuously according to the chromatographic conditions in Example 1, and obtain 6 chromatograms. Calculate the peak area and retention time of each characteristic peak, substitute into the regression equation to calculate the content of each index component, and calculate the RSD value. The results are shown in Table 14 and Table 15.

The retention time of each characteristic peak precision test of table 14

Table 15 Content of each characteristic peak precision test (μg·mL ^-1 )

It can be seen from Table 14 and Table 15 that the RSD values of the retention time of each characteristic peak are all <3%, and the RSD values of the content are all <3%, which shows that the precision of this method is good.

2.3 Repeatability test

Get Lanqin Oral Liquid (17050232), according to the preparation method of the test solution in Example 1, prepare 6 parts of the test solution in parallel, inject samples respectively according to the chromatographic conditions in Example 1, and obtain 6 chromatograms. Calculate the peak area and retention time of each characteristic peak, substitute into the regression equation to calculate the content of each index component, and calculate the RSD value. The results are shown in Table 16 and Table 17.

The retention time of each characteristic peak repeatability test of table 16

Table 17 The content of each characteristic peak repeatability test (μg·mL ^-1 )

It can be seen from Table 16 and Table 17 that the RSD values of the retention time of each characteristic peak are all <3%, and the RSD values of the content are all <3%, which shows that the repeatability of the method is good.

2.4 Stability test

Get Lanqin Oral Liquid (17050232), prepare 6 parts of need testing solutions in parallel according to the preparation method of the need testing solution of Example 1, and press the chromatographic conditions in Example 1, respectively at 0h, 2h, 4h, 8h, 12h , 24h condition injection, get 6 chromatograms. Calculate the peak area and retention time of each common characteristic peak, and substitute into the regression equation to calculate the content of each index component. The results are shown in Table 18 and Table 19.

The retention time of each characteristic peak stability test of table 18

Table 19 Content of each characteristic peak stability test (μg·mL ^-1 )

It can be seen from Table 18 and Table 19 that the RSD values of the retention time of each characteristic peak are all <3%, and the RSD values of the content are <3%, indicating that the characteristic components have good stability within 24 hours at room temperature.

2.5 Adding sample stability test

Take by weighing the same batch of Lanqin Oral Liquid, prepare 9 parts of need testing solution by the preparation method of the need testing solution of embodiment 1, add precision respectively containing adenosine, Yi Chun, phellodine hydrochloride, chlorogenic acid, Magnolin, geniposide, benzoic acid, berberine hydrochloride, wogonin, baicalein, and wogonin reference substance solutions were injected in appropriate amounts according to the chromatographic conditions in Example 1, and 9 chromatograms were obtained. Calculate the average recovery rate and RSD value of each index chemical composition. The results are shown in Table 20-Table 31.

The sample recovery rate result of table 20 adenosine

It can be seen from Table 20 that the result of the sample recovery of adenosine meets the test requirements.

Table 21 shows the results of the sample recovery rate of Gao Yichun

From Table 21, it can be seen that the recovery rate of the addition of Yichun in Table 21 meets the test requirements.

The sample addition recovery result of table 22 phellodine hydrochloride

As can be seen from Table 22, the addition recovery rate result of Phellodendron hydrochloride meets the test requirements

The sample recovery rate result of table 23 chlorogenic acid

It can be seen from Table 23 that the sample recovery of chlorogenic acid meets the test requirements.

The sample addition recovery rate result of table 24 Magnolin

As can be seen from Table 24, the sample recovery result of magnolanine meets the test requirements.

The sample recovery rate result of table 25 geniposide

It can be seen from Table 25 that the recovery rate of geniposide met the test requirements.

The sample addition recovery rate result of table 26 benzoic acid

As can be seen from Table 26, the sample recovery result of benzoic acid meets the test requirements.

The sample addition recovery rate result of table 27 berberine hydrochloride

As can be seen from Table 27, the sample recovery result of berberine hydrochloride meets the test requirements.

The sample recovery rate result of table 28 baicalin

It can be seen from Table 28 that the recovery rate of baicalin meets the test requirements.

The sample recovery rate result of table 29 wogonoside

It can be seen from Table 29 that the recovery rate of wogonoside meets the test requirements.

The sample recovery rate result of table 30 baicalein

It can be seen from Table 30 that the recovery rate of baicalein meets the test requirements.

The sample recovery rate result of table 31 wogonin

It can be seen from Table 31 that the recovery rate of wogonin meets the test requirements.

3. Determination of content of index components

Get 19 different batches of Lanqin oral liquid of above table 1, prepare 19 parts of need testing solution by the preparation method of need testing solution of embodiment 1, inject samples respectively by chromatographic conditions in embodiment 1, record each index composition The peak area was substituted into the linear regression equation to calculate the content, each sample was measured twice, and the average value was obtained. Calculate the content of each index chemical component in different batches of Lanqin Oral Liquid. The results are shown in Table 32.

It can be seen from Table 32 that among different batches of Lanqin Oral Liquid finished preparations, there are no significant differences in the contents of the 12 efficacy index components except for some index components. It proves that the quality of Lanqin Oral Liquid is uniform and stable.

Example 3 Establishment of Fingerprints of Lanqin Oral Liquid Key Process Intermediates

1. Fingerprint of Concentration Process Intermediate I

Take an appropriate amount of 9 different batches of Lanqin concentrated process intermediate I in the above table 2, add 60% methanol aqueous solution to dilute, mix well, and pass through a 0.45 μm microporous membrane as the test solution. Sample injection by chromatographic conditions in Example 1, obtain the liquid chromatography of need testing solution. Import the liquid chromatogram into the "Chromatographic Fingerprint Similarity Evaluation System 2004A Edition" compiled by the National Pharmacopoeia Committee to process the chromatogram data. Obtain the overlay chromatogram (see Fig. 20) and control spectrum (see Fig. 21) of enrichment process intermediate I, carry out similarity analysis to the fingerprint spectrum of 9 batches of Lanqin concentration process intermediate I and the control fingerprint spectrum generated, the results are shown in Table 33.

Table 33 Similarity of Fingerprints of Nine Batches of Lanqin Concentration Process Intermediate I

It can be seen from Table 33 that the similarity between the fingerprints of different batches of concentrated intermediate I and the control spectrum is between 0.990 and 1, indicating that the similarity between different batches is high and the process is relatively stable.

2. The fingerprint of the intermediate II of the cream collection process

Take the appropriate amount of Lanqin Harvesting Process Intermediate II from 9 different batches in Table 3 above, add 60% methanol aqueous solution to dilute, mix well, and pass through a 0.45 μm microporous membrane as the test solution. Sample injection by chromatographic conditions in Example 1, obtain the liquid chromatography of need testing solution. Import the liquid chromatogram into the "Chromatographic Fingerprint Similarity Evaluation System 2004A Edition" compiled by the National Pharmacopoeia Committee to process the chromatogram data. Obtain the overlay chromatogram (see Fig. 22) and the control spectrum (see Fig. 23) of the process intermediate II of the ointment harvesting process, and carry out similarity analysis between the fingerprints of the intermediate II of the process intermediate II of the harvesting ointment and the generated control fingerprints, The results are shown in Table 34.

Table 34 Similarity of Fingerprints of 9 Batches of Lanqin Harvesting Ointment Process Intermediate II

It can be seen from Table 34 that the similarity between different batches of cream collection process intermediate II fingerprints and the control spectrum is between 0.992 and 1, indicating that the similarity between different batches is high and the process is relatively stable.

3. The fingerprint of the initial preparation process intermediate III

Take an appropriate amount of the 6 different batches of blue baicalensis primary compounding process intermediate III in the above table 4, add 60% methanol solution to dilute, mix well, pass through a 0.45 μm microporous membrane, and use it as the test solution. Sample injection by chromatographic conditions in Example 1, obtain the liquid chromatography of need testing solution. Import the liquid chromatogram into the "Chromatographic Fingerprint Similarity Evaluation System 2004A Edition" compiled by the National Pharmacopoeia Committee to process the chromatogram data. Obtain the overlay chromatogram (see Fig. 24) and the control spectrum (see Fig. 25) of the primary preparation process intermediate III, and carry out similarity analysis between the fingerprints of the initial preparation process intermediate III of 6 batches of Scutellaria baicalensis and the generated control fingerprints, The results are shown in Table 35.

Table 35 Similarity of Fingerprints of Six Batches of Lanqin Primary Compounding Process Intermediate III

It can be seen from Table 35 that the similarity between the fingerprints of different batches of initial preparation process intermediate III and the control spectrum is between 0.999 and 1, indicating that the similarity between different batches is high and the process is relatively stable.

4.Fingerprint of fine compounding process intermediate IV

Take an appropriate amount of the middle IV of the 6 different batches of Lanqin essence in the above table 5, add 60% methanol aqueous solution to dilute, mix well, pass through a 0.45 μm microporous membrane, and use it as the test solution. Sample injection by chromatographic conditions in Example 1, obtain the liquid chromatography of need testing solution. Import the liquid chromatogram into the "Chromatographic Fingerprint Similarity Evaluation System 2004A Edition" compiled by the National Pharmacopoeia Committee to process the chromatogram data. Obtain the overlay chromatogram (see Figure 26) and the control spectrum (see Figure 27) of fine compounding process intermediate IV, and carry out similarity analysis on the fingerprints of 6 batches of Lanqin fine compounding process intermediate IV and the generated control fingerprints, The results are shown in Table 36.

Table 37 Similarity of Fingerprints of 6 Batches of Lanqin Jing Compounding Process Intermediate IV

It can be seen from Table 36 that the similarity between the IV fingerprints of different batches of precision blending process intermediates and the control spectrum is between 0.999 and 1, indicating that the similarity between different batches is high and the process is relatively stable.

Embodiment 4 Lanqin Oral Liquid Key Process Intermediate Drug Effect Index Components Changes

Get the appropriate amount of each key process intermediate of Lanqin Oral Liquid, make the solution of the test product, inject and detect by the chromatographic conditions in Example 1, measure and record each drug effect index component in each key process of preparation production under the wavelength 230nm and 276nm The peak area of the intermediate, the results are shown in Table 37. Investigate the content change trend and degree of each medicinal ingredient (see Figure 28-Figure 29).

Table 37 The peak area changes of the 12 pharmacodynamic index components during the preparation process

From the results, it can be known that the variation of each component in different technological processes is as follows: from the concentration process to the extraction process, except for a slight increase in the content of adenosine, the content of other components all showed a significant decline; from the extraction process to the initial extraction process In the compounding process, it was reported that the content of Yichun dropped sharply, the contents of Phellodendronine hydrochloride, chlorogenic acid, geniposide, adenosine, and wogonin increased, and the contents of magnolialine, baicalin, wogonin, and baicalein increased slightly , the content of benzoic acid increased sharply and significantly, and the content of berberine hydrochloride showed a slight downward trend; from the initial blending link to the fine blending link, the contents of the 12 efficacy index components all had a downward trend in varying degrees; from the fine blending link When it comes to the final product of the preparation, it is reported that the content of Yichun has increased sharply and the content of magnolialine has increased; Phellodendron hydrochloride, chlorogenic acid, geniposide, benzoic acid, berberine hydrochloride, adenosine, baicalin, and Scutellaria baicalensis The contents of glycosides, baicalein and wogonin all decreased in varying degrees. Based on a single drug efficacy index component, the change rule of its content affected by the process is as follows: the content of Yichun showed a sharp decline in the three stages of concentration, ointment collection, and initial preparation, and the content of the fine preparation liquid changed. Not much, but the content of the final product showed a sharp rise from the fine blending to the content of the concentrated solution, which shows that the steps of extracting the cream, the initial blending link and the final product link are the key process steps for the content control of Yichun. The content of phellodine hydrochloride decreased significantly from the concentration process to the ointment collection process, and the content of other process links did not change much, so the content of phellodine hydrochloride should be controlled during the ointment collection process. Except for the slight increase in the content of chlorogenic acid in the initial compounding process, the rest of the process processes showed a downward trend; The decline trend of the content in the alcohol precipitation and ointment collection process is obvious, which should be the key control step, and the relative content of other process links has little change; the content of benzoic acid in the concentrated solution and the ointment collection is relatively low. The content of adenosine increased sharply, and then its content decreased slightly during the process; the content of adenosine reached the maximum in the initial preparation process, and its content in the fine preparation and final product decreased slightly, which was not much different from the content in the concentrated solution and the harvested ointment.

The above test results show that a method for establishing the fingerprint of Lanqin Oral Liquid and its fingerprints provided by the present invention can comprehensively analyze the overall components of Lanqin Oral Liquid, and can basically represent and reflect the active ingredients and substances of Lanqin Oral Liquid Basically, by establishing the fingerprints of Lanqin Oral Liquid and its process intermediates, the content and variation of each index component of the oral liquid in different process processes can be detected, which has certain identification significance for the quality control of different process links of the preparation. It provides a certain theoretical basis and support for the establishment of the whole process quality control system, which can comprehensively reflect the quality information of Lanqin Oral Liquid and ensure the uniform and stable product quality. The research found that: 1. The 26 common peaks in the fingerprint of Lanqin Oral Liquid basically found a clear attribution in the chromatograms of the positive control and negative control of the four medicinal materials of Radix Isatidis, Scutellaria, Gardenia, and Cortex Phellodendri. It can be seen that peaks 1 and 4 in the common peaks of the fingerprints come from Radix Radix Radix; peaks 12, 18, 19, 20, 21, 22, 23, 24, 25, and 26 in the common peaks come from Scutellaria baicalensis; , Peaks 7, 8, 10, 11, and 16 come from Cortex Phellodendri; peaks 2, 3, 6, 9, 15, and 17 in the common peaks come from Gardenia; peaks 13 and 14 exist in Scutellaria baicalensis and Gardenia In the medicinal materials. It can be seen that the fingerprint of Lanqin Oral Liquid can basically represent the material basis of Lanqin Oral Liquid. 2. The content of the 12 kinds of drug efficacy index components in each process intermediate has a different trend and degree of change compared with Lanqin Oral Liquid, but they all have obvious content changes, so the corresponding key should be formulated according to specific changes. Point quality control standard. The content of most of the index components in the concentrated solution and the initial preparation solution increased significantly, and there was no significant difference in the content of most of the index components in the ointment collection liquid and the fine preparation solution. Therefore, the concentration link and the initial preparation link are key process control links and should be Establish quality control standards for key points to ensure that the quality of traditional Chinese medicine preparations is controlled from the intermediate link.

Although the embodiments disclosed in the present application are as above, the content described is only the embodiments adopted to facilitate understanding of the present application, and is not intended to limit the present application. Anyone skilled in the field of this application can make any modifications and changes in the form and details of implementation without departing from the spirit and scope disclosed in this application, but the scope of patent protection of this application must still be The scope defined by the appended claims shall prevail.

Claims (11)

1. a kind of method for building up of Lanqin oral liquid finger-print, the method for building up the following steps are included:

(1) test solution the preparation of test solution: is prepared into Lanqin oral liquid；

(2) preparation of reference substance solution: dissolved with adenosine reference substance, epigoitrin reference substance, hydrochloric acid phellodendrine reference substance, green Ortho acid reference substance, magnoflorine reference substance, Gardenoside reference substance, benzoic acid reference substance, Berberine hydrochloride reference substance, scutelloside pair Solution according to product, wogonoside reference substance, baicalein reference substance and wogonin reference substance is reference substance solution；

(3) using the test solution of HPLC difference determination step (1) and the reference substance solution of step (2), and institute is respectively obtained State the liquid chromatogram of test solution and the reference substance solution；

(4) liquid chromatogram for obtaining step (3) imports similarity evaluation analysis, obtains blue a kind of reed mentioned in ancient books The finger-print of oral solution.

2. the method for building up of Lanqin oral liquid finger-print according to claim 1, wherein the step (1) for examination The preparation of product solution includes: accurate 100~300 μ l of Lanqin oral liquid that draws in 10mL centrifuge tube, and the volume of 4~8mL is added Score is that the methanol aqueous solution of 50%-100% is diluted, and 0.45 μm of miillpore filter is crossed after mixing；

Alternatively, the preparation of the test solution of the step (1) includes: that the accurate 200 μ l of Lanqin oral liquid that draws is centrifuged in 10mL Guan Zhong, the methanol that 4.8mL is added are diluted, and 0.45 μm of miillpore filter is crossed after mixing.

3. the method for building up of Lanqin oral liquid finger-print according to claim 1, wherein the control of the step (2) The preparation of product solution includes: that precision weighs adenosine reference substance, epigoitrin reference substance, hydrochloric acid phellodendrine reference substance, chlorogenic acid pair According to product, magnoflorine reference substance, Gardenoside reference substance, benzoic acid reference substance, Berberine hydrochloride reference substance, scutelloside reference substance, The methanol-water that volume fraction is 70%-100% is added in wogonoside reference substance, baicalein reference substance and wogonin reference substance Solution is made every 1mL and contains 5~8 μ g adenosines, 2~6 μ g epigoitrins, 5~8 μ g hydrochloric acid phellodendrines, 30~50 μ g chlorogenic acids, 2 ~5 μ g magnoflorines, 200~300 μ g Gardenosides, 100~200 μ g benzoic acid, 2~5 μ g Berberine hydrochlorides, 150~250 μ g are yellow The reference substance solution of a kind of reed mentioned in ancient books glycosides, 50~100 μ g wogonosides, 8~15 μ g baicaleins, 8~15 μ g wogonins；

Alternatively, the preparation of step (2) reference substance solution includes: to take scutelloside reference substance appropriate, and it is accurately weighed, add volume point Number is that 70% methanol aqueous solution dissolves, and being configured to content is 208 μ gmL^-1Reference substance solution, take adenosine reference substance, table respectively It accuses according to spring reference substance, hydrochloric acid phellodendrine reference substance, chlorogenic acid reference substance, magnoflorine reference substance, Gardenoside reference substance, benzoic acid Reference substance, Berberine hydrochloride reference substance, wogonoside reference substance, baicalein reference substance, wogonin reference substance are appropriate, and precision claims It is fixed, add methanol to dissolve, being configured to content is respectively 7.52 μ gmL^-1、3.84μg·mL^-1、6.44μg·mL^-1、32.96μg· mL^-1、2.52μg·mL^-1、284.00μg·mL^-1、162.00μg·mL^-1、4.56μg·mL^-1、98.00μg·mL^-1、10.60μ g·mL^-1、9.76μg·mL^-1Reference substance solution.

4. the method for building up of Lanqin oral liquid finger-print according to claim 1, wherein the step (3) includes setting Set HPLC chromatogram condition: chromatographic column uses octadecylsilane chemically bonded silica for filler, and 25-35 DEG C of column temperature, flow velocity 0.8- 1.2mL/min, Detection wavelength 250-320nm, and open DAD detection；It is 0.06- with volume fraction using acetonitrile as mobile phase A 0.2% phosphate aqueous solution is Mobile phase B, gradient elution in the range of volume ratio is 5~95:100~0；

It is accurate respectively to draw 5~20 μ L of test solution and control solution, high performance liquid chromatograph is injected, is measured, respectively To the liquid chromatogram of test solution and control solution.

5. the method for building up of Lanqin oral liquid finger-print according to claim 4, wherein the step (3) includes setting Set HPLC chromatogram condition: chromatographic column uses octadecylsilane chemically bonded silica for filler, with 2695 ACQUITY of Waters HPLC^TM(4.6mm × 250mm, 5.0 μm) be chromatographic column, 30 DEG C of column temperature, flow velocity 1.0mL/min, Detection wavelength 230nm, and Open DAD detection；It is 0.2% phosphate aqueous solution as Mobile phase B using volume fraction using acetonitrile as mobile phase A, carries out gradient and wash De-, mobile phase A, the variation of the ratio of B are 0~12min, and A:B is 5%:95% → 11%:89%；12~24min, A:B are 11%:89% → 11%:89%；24~38min, A:B are 11%:89% → 23%:77%；38~50min, A:B 23%: 77% → 28%:72%；50~55min, A:B are 28%:72% → 31%:69%；55~70min, A:B 31%:69% → 100%:0%；

It is accurate respectively to draw 10 μ L of test solution and control solution, high performance liquid chromatograph is injected, measurement respectively obtains confession The liquid chromatogram of test sample solution and reference substance solution.

6. the method for building up of Lanqin oral liquid finger-print according to any one of claims 1-5, wherein the step (4) include: using similarity evaluation 2004 A editions, the liquid chromatogram that step (3) is obtained Data importing, Supplements, Data Matching, analysis obtain the finger-print of Lanqin oral liquid.

7. the method for building up of Lanqin oral liquid finger-print according to claim 6, wherein what the step (4) obtained Lanqin oral liquid finger-print includes that corresponding No. 1 peak of adenosine, corresponding No. 4 peaks of epigoitrin, hydrochloric acid phellodendrine are No. 5 corresponding Corresponding No. 6 peaks of peak, chlorogenic acid, corresponding No. 7 peaks of magnoflorine, corresponding No. 9 peaks of Gardenoside, benzoic acid are No. 15 corresponding Corresponding No. 16 peaks of peak, Berberine hydrochloride, corresponding No. 17 peaks of scutelloside, corresponding No. 22 peaks of wogonoside, baicalein are corresponding No. 24 peaks and corresponding No. 25 peaks of wogonin, relative retention time is respectively 0.120,0.587,0.711,0.777, 0.806,1.000,1.882,1.946,2.002,2.615,2.856 and 3.003.

8. the method for building up of Lanqin oral liquid finger-print according to claim 7, wherein what the step (4) obtained The shared peak of Lanqin oral liquid finger-print further includes that No. 2 peaks, the relative retention times that relative retention time is 0.382 are 0.453 No. 3 peaks, No. 8 peaks that relative retention time is 0.846, No. 10 peaks that relative retention time is 1.400, opposite reservation No. 11 peaks that time is 1.499, No. 12 peaks that relative retention time is 1.731, No. 13 peaks that relative retention time is 1.812, No. 14 peaks that relative retention time is 1.847, No. 18 peaks that relative retention time is 227,19 that relative retention time is 2.375 Number peak, No. 20 peaks that relative retention time is 2.414, No. 21 peaks, the relative retention times that relative retention time is 2.495 are 2.835 No. 23 peaks, No. 26 peaks that relative retention time is 3.015.

9. the method for building up of Lanqin oral liquid finger-print according to claim 1, wherein the Lanqin oral liquid can be with It is replaced with Lanqin oral liquid critical process intermediate；The Lanqin oral liquid critical process intermediate includes concentration technology intermediate I, cream technique intermediate II is received, just with technique intermediate III, essence with technique intermediate compound IV.

10. the obtained finger-print of method for building up of Lanqin oral liquid finger-print of any of claims 1-9.

11. finger-print described in any one of claim 10 is in Lanqin oral liquid overall process quality evaluation system or Lanqin oral liquid work The application of skill key point control.